CYP3A4 and microRNA-122 are involved in the apoptosis of HepG2 cells induced by ILs 1-decyl-3-methylimidazolium bromide.

Ionic liquids (ILs) as green alternatives for volatile organic solvents are increasingly used in commercial applications. It is necessary to explore the cytotoxic mechanism of ILs to reduce the risk to human health. For this purpose, cell viability, apoptosis, cytochrome P450 3A4 (CYP3A4), glucose transporter type 2 (GLUT2), and microRNA-122 (miR-122) gene expression in HepG2 cells was evaluated after IL exposure. The results showed that ILs reduced the viability of HepG2 cells through apoptotic cell death. Moreover, ILs markedly upregulated the transcription and protein levels of CYP3A4, but did not affect the expression of GLUT2 in either messenger RNA level or protein level. Finally, ILs increased the expression of miR-122 and inhibition of miR-122 with miR-122 inhibitor blocked ILs-induced apoptosis in HepG2 cells. This finding may contribute to an increased understanding of the in vitro molecular toxicity mechanism of ILs to further understand IL-related human health risks.

Leucothrix sargassi sp. nov., isolated from a marine alga [Sargassum natans (L.) Gaillon].

A novel bacterial strain, C3212T, was isolated from a marine alga collected from the sea shore of Yantai, China. The strain was Gram-stain-negative, rod-shaped, aerobic, non-motile, and oxidase- and catalase-positive. Growth was observed at 8-37 °C (optimum, 28 °C), at pH 6.0-9.0 (optimum, pH 7.0) and in the presence of 1.0-7.0 % (w/v) NaCl (optimum, 4.0 %). The major respiratory quinone was ubiquinone-8 (Q-8). The polar lipids of strain C3212T consisted of diphosphatidylglycerol (cardiolipin), phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminophospholipid, an unidentified phospholipid and an unidentified polar lipid. The major fatty acids were C16 : 1ω6c and/or C16 : 1ω7c, and C18 : 1ω6c and/or C18 : 1ω7c. The DNA G+C content of strain C3212T was 44.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was related most closely to Leucothrix pacifica XH122T, Leucothrix arctica IMCC 9719T and Leucothrix mucor DSM 2157T with similarities of 98.0, 97.5 and 94.3 %, respectively. Estimated DNA-DNA hybridization values were 14.2, 20.7 and 13.9 % between strain C3212T and L. pacifica XH122T, L. arctica IMCC 9719T and L. mucor DSM 2157T, respectively. Phenotypic, phylogenetic and genomic analyses revealed that strain C3212T represents a novel species of the genus Leucothrix, for which the name Leucothrix sargassi sp. nov. is proposed. The type strain is C3212T (=MCCC 1K03600T=KCTC 72121T).

Shewanella gelidii sp. nov., isolated from the red algae Gelidium amansii, and emended description of Shewanella waksmanii.

A novel Gram-stain-negative, straight or slightly curved rod-shaped, non-spore-forming, facultatively anaerobic bacterium with a single polar flagellum, designated RZB5-4T, was isolated from a sample of the red algae Gelidium amansii collected from the coastal region of Rizhao, PR China (119.625° E 35.517° N). The organism grew optimally between 24 and 28 °C, at pH 7.0 and in the presence of 2-3 % (w/v) NaCl. The strain required seawater or artificial seawater for growth, and NaCl alone did not support growth. Strain RZB5-4T contained C16 : 1ω7c and/or C16 : 1ω6c, C16 : 0 and iso-C15 : 0 as the dominant fatty acids. The respiratory quinones detected in strain RZB5-4T were ubiquinone 7, ubiquinone 8, menaquinone 7 and methylmenaquinone 7. The polar lipids of strain RZB5-4T comprised phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, one unidentified glycolipid, one unidentified phospholipid and one unknown lipid. The DNA G+C content of strain RZB5-4T was 47 mol %. Phylogenetic analysis based on 16S rRNA and gyrase B (gyrB) gene sequences showed that strain RZB5-4T belonged to the genus Shewanella, clustering with Shewanella waksmanii ATCC BAA-643T. Strain RZB5-4T exhibited the highest 16S rRNA gene sequence similarity value (96.6 %) and the highest gyrB gene sequence similarity value (80.7 %), respectively, to S. waksmanii ATCC BAA-643T. On the basis of polyphasic analyses, strain RZB5-4T represents a novel species of the genus Shewanella, for which the name Shewanella gelidii sp. nov. is proposed. The type strain is RZB5-4T (=JCM 30804T=KCTC 42663T=MCCC 1K00697T).

Pseudofulvibacter marinus sp. nov., isolated from seawater.

A novel Gram-stain-negative, rod-shaped, non-spore-forming, non-flagellated, strictly aerobic strain, designated RZW2-1T, was isolated from coastal seawater of the Yellow Sea in China (35.475° N 119.613° E). The organism grew optimally at 24 °C, at pH 7.0 and in the presence of 3.0 % (w/v) NaCl. The strain requires seawater or artificial seawater for growth and NaCl alone does not support growth. Strain RZW2-1T contained MK-6 as the only respiratory quinone and iso-C15 : 0, iso-C15 : 1 G and 10-methyl C16 : 0 and/or iso-C17 : 1ω9c as the dominant fatty acids. The polar lipids of strain RZW2-1T were four unidentified phospholipids (PL1-PL4), two unknown lipids (L1, L2) and one unidentified aminolipid (AL1). The DNA G+C content of strain RZW2-1T was 32 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was most closely related to the type strain of the only described species of genus Pseudofulvibacter, Pseudofulvibacter geojedonensis YCS-9T, with 95.1 % 16S rRNA gene sequence similarity. On the basis of polyphasic analyses, strain RZW2-1T represents a novel species of the genus Pseudofulvibacter, for which the name Pseudofulvibacter marinus sp. nov. is proposed. The type strain is RZW2-1T ( = JCM 30826T = MCCC 1K00695T).

Estimation of the cool executive function using frontal electroencephalogram signals in first-episode schizophrenia patients.

BACKGROUND: In schizophrenia, executive dysfunction is the most critical cognitive impairment, and is associated with abnormal neural activities, especially in the frontal lobes. Complexity estimation using electroencephalogram (EEG) recording based on nonlinear dynamics and task performance tests have been widely used to estimate executive dysfunction in schizophrenia. METHODS: The present study estimated the cool executive function based on fractal dimension (FD) values of EEG data recorded from first-episode schizophrenia patients and healthy controls during the performance of three cool executive function tasks, namely, the Trail Making Test-A (TMT-A), Trail Making Test-B (TMT-B), and Tower of Hanoi tasks. RESULTS: The results show that the complexity of the frontal EEG signals that were measured using FD was different in first-episode schizophrenia patients during the manipulation of executive function. However, no differences between patients and controls were found in the FD values of the EEG data that was recorded during the performance of the Tower of Hanoi task. CONCLUSIONS: These results suggest that cool executive function exhibits little impairment in first-episode schizophrenia patients.

Effect of 1-octyl-3-methylimidazolium chloride on cell replication and membrane permeability of Escherichia coli DH5α.

Ionic liquids (ILs) have elicited attention due to their unique properties. ILs may pose environmental risks to aquatic ecosystems once released into water during generation and application. Therefore, the toxic and antimicrobial properties of ILs should be analysed. This study aims to evaluate the cytotoxicity of 1-octyl-3-methylimidazolium chloride ([C8mim] [Cl]) on Escherichia coli DH5α by MTT (3-[4,5-dimethylthiazol-2yl]-2,5 diphenyl tetrazolium bromide) assay, and to determine the effect of [C8mim] [Cl] on the replication and membrane permeability of E. coli DH5α. The results showed that [C8mim] [Cl] decreased cell viability and inhibited bacterial cell replication. [C8mim] [Cl] increased protein and nucleic acid contents in the extracellular fluid, damaged the cell membrane, and increased membrane permeability. The increase of cell membrane permeability induced by [C8mim] [Cl] could be the cause of decreased cell viability and replication.

[3D bioprinting: classification, evaluation, and application in vascular tissue engineering].

Cardiovascular diseases are major diseases, and there is lack of artificial blood vessels with small diameters which can be applied in coronary artery bypass surgery. The conventional vascular scaffold preparation techniques in tissue engineering have shortcomings in regulating the diameter, geometric shape, and interconnectivity of the scaffold. 3D bioprinting can simulate the natural structure of the vascular tissue, accurately print live cells and biomaterials, and regulate the microstructure and porosity of scaffolds on the nanoscale, providing new ideas for vascular tissue engineering. This article systematically evaluates the classification of 3D bioprinting technologies and reviews the latest research progress of 3D bioprinting in vascular tissue engineering. It summarizes the advantages of 3D bioprinting and points out the problems that need to be solved, such as the immune rejection of blood vessel materials, providing reference for the further research.

Tumor microenvironment-responsive size-changeable and biodegradable HA-CuS/MnO2 nanosheets for MR imaging and synergistic chemodynamic therapy/phototherapy.

Tumor microenvironment (TME)-responsive size-changeable and biodegradable nanoplatforms for multimodal therapy possess huge advantages in anti-tumor therapy. Hence, we developed a hyaluronic acid (HA) modified CuS/MnO2 nanosheets (HCMNs) as a multifunctional nanoplatform for synergistic chemodynamic therapy (CDT)/photothermal therapy (PTT)/photodynamic therapy (PDT). The prepared HCMNs exhibited significant NIR light absorption and photothermal conversion efficiency because of the densely deposited ultra-small sized CuS nanoparticles on the surface of MnO2 nanosheet. They could precisely target the tumor cells and rapidly decomposed into small sized nanostructures in the TME, and then efficiently promote intracellular ROS generation through a series of cascade reactions. Moreover, the local temperature elevation induced by photothermal effect also promote the PDT based on CuS nanoparticles and the Fenton-like reaction of Mn2+, thereby enhancing the therapeutic efficiency. Furthermore, the T1-weighted magnetic resonance (MR) imaging was significantly enhanced by the abundant Mn2+ ions from the decomposition process of HCMNs. In addition, the CDT/PTT/PDT synergistic therapy using a single NIR light source exhibited considerable anti-tumor effect via in vitro cell test. Therefore, the developed HCMNs will provide great potential for MR imaging and multimodal synergistic cancer therapy.

Responses of the antioxidant system in QGY-7701 cells to the cytotoxicity and apoptosis induced by 1-octyl-3-methylimidazolium chloride.

This study aims to evaluate the cytotoxicity and responses of the cellular antioxidant system of 1-octyl-3-methylimidazolium chloride ([C8 mim][Cl]) on human hepatocarcinoma QGY-7701 cells. The results show that [C8 mim][Cl] can inhibit QGY-7701 cell growth and decrease their viabilities in a dose-dependent manner. The results also reveal that [C8 mim][Cl] exposure can induce apoptosis in the [C8 mim][Cl]-treated QGY-7701 cells. In addition, the results of biochemical assays show that [C8 mim][Cl] exposure causes overproduction of reactive oxygen species (ROS), inhibits superoxide dismutase (SOD) and catalase (CAT) activities, decreases reduced glutathione (GSH) content, and increases the cellular malondialdehyde (MDA) level. These results suggest that ROS-mediated oxidative stress may be responsible for the apoptosis induced by [C8 mim][Cl] in QGY-7701 cells.

Construction of spider silk protein small-caliber tissue engineering vascular grafts based on dynamic culture and its performance evaluation.

Tissue engineering is an alternative method for preparing small-caliber (<6 mm) vascular grafts. Dynamic mechanical conditioning is being researched as a method to improve mechanical properties of tissue engineered blood vessels. This method attempts to induce unique reaction in implanted cells that regenerate the matrix around them, thereby improving the overall mechanical stability of the grafts. In this study, we used a bioreactor to seed endothelial cells and smooth muscle cells into the inner and outer layers of the electrospun spider silk protein scaffold respectively to construct vascular grafts. The cell proliferation, mechanical properties, blood compatibility and other indicators of the vascular grafts were characterized in vitro. Furthermore, the vascular grafts were implanted in Sprague Dawley rats, and the vascular grafts' patency, extracellular matrix formation, and inflammatory response were evaluated in vivo. We aimed to construct spider silk protein vascular grafts with the potential for in vivo implantation by using a pulsating flow bioreactor. The results showed that, when compared with the static culture condition, the dynamic culture condition improved cell proliferation on vascular scaffolds and enhanced mechanical function of vascular scaffolds. In vivo experiments also showed that the dynamic culture of vascular grafts was more beneficial for the extracellular matrix deposition and anti-thrombogenesis, as well as reducing the inflammatory response of vascular grafts. In conclusion, dynamic mechanical conditioning aid in the resolution of challenges impeding the application of electrospun scaffolds and have the potential to construct small-caliber blood vessels with regenerative function for cardiovascular tissue repair.

Apoptosis caused by imidazolium-based ionic liquids in PC12 cells.

The cytotoxicity of alkylmethylimidazolium-based ionic liquids on rat pheochromocytoma (PC12) cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase release (LDH), and acridine orange staining in the present study. Mitochondrial depolarization, DNA fragmentation, reactive oxygen species (ROS) levels, and caspase-3 activity were also determined. The results showed dose-dependent cytotoxicity of ionic liquids on PC12 cells, and the ionic liquids with longer lateral chains had stronger cytotoxicity. Additionally, we found that exposure to the ionic liquid 1-octyl-3-methylimidazolium bromide ([C(8)mim]Br) provoked cellular LDH release, increased mitochondrial depolarization, induced cellular transmogrification, nuclear shrinkage and DNA fragmentation, and promoted caspase-3 activity and ROS levels in PC12 cells. These results suggest that [C(8)mim]Br may induce PC12 cell apoptosis triggered by excessive ROS and mediated by mitochondrial depolarization and permeability transition. Our result may be helpful for illuminating the cytotoxicity mechanism of alkylmethylimidazolium-based ionic liquids and safely using them in the future.

Progress of Transposon Vector System for Production of Recombinant Therapeutic Proteins in Mammalian Cells.

In recent years, mammalian cells have become the primary host cells for the production of recombinant therapeutic proteins (RTPs). Despite that the expression of RTPs in mammalian cells can be improved by directly optimizing or engineering the expression vectors, it is still influenced by the low stability and efficiency of gene integration. Transposons are mobile genetic elements that can be inserted and cleaved within the genome and can change their inserting position. The transposon vector system can be applied to establish a stable pool of cells with high efficiency in RTPs production through facilitating the integration of gene of interest into transcriptionally active sites under screening pressure. Here, the structure and optimization of transposon vector system and its application in expressing RTPs at high level in mammalian cells are reviewed.

Improved hemocompatibility by modifying acellular blood vessels with bivalirudin and its biocompatibility evaluation.

The incidence rate of cardiovascular diseases is increasing year by year. The demand for coronary artery bypass grafting has been very large. Acellular blood vessels have potential clinical application because of their natural vascular basis, but their biocompatibility and anticoagulant energy need to be improved. We decellularized the abdominal aorta of SD rats, and then modified with bivalirudin via polydopamine. The mechanical properties, blood compatibility, cytocompatibility, immune response, and anticoagulant properties were evaluated, and then the bivalirudin-modified acellular blood vessels were implanted into rats for remodeling evaluation in vivo. The results we got show that the bivalirudin-modified acellular blood vessels showed good cytocompatibility and blood compatibility, and its anti-inflammatory trend was dominant in the immune response. After 3 months of transplantation, the bivalirudin-modified acellular blood vessels did not easily form thrombus. It was not easy to form calcification and could make the host cells grow better. Through vascular stimulation and immunofluorescence test, we found that vascular smooth muscle cells and endothelial cells proliferated well in the bivalirudin group. Bivalirudin-modified acellular blood vessels provided new idea for small diameter tissue engineering blood vessels, and may become a potential clinical substitute for small-diameter vascular grafts.

Positional effects of the matrix attachment region on transgene expression in stably transfected CHO cells.

Previous work has shown that the MAR (matrix attachment region) could increase transgene expression in stably transfected CHO (Chinese-hamster ovary) cells. To study the positional effect of MAR on transgene expression, three expression vectors were constructed which contained the human beta-globin MAR in different sites, including the vector with two MARs flanking the CAT (chloramphenicol acetyltransferase) expression cassette, one MAR at the 59 or 39 site. These vectors were transfected into CHO cells. The level of CAT gene expression was most effectively increased by two MARs flanking the CAT expression cassette. This increase was also seen when MAR was inserted at the 59 site upstream of the expression cassette, whereas the transgene expression level decreased when MAR was inserted at the 39 site downstream of the expression cassette. We have also shown that the transgene expression level is not directly proportional to the gene copy number, and gene copy number dependency does not exist.

Nucleotide sequence and expression of the 14-3-3 from the halotolerant alga Dunaliella salina.

Previously we reported the nucleotide sequence of a 14-3-3 cDNA cloned from the unicellular green alga Dunaliella salina, however, the nucleotide sequence of this gene have not been reported so far. In the present study, the cloning and characterization of the nucleotide sequence, the gene copy and expression were undertaken. The coding sequence of the gene was found to be interrupted by five introns of 132, 266, 153, 152 and 625 bp, respectively. Introns 3-5 were found in conserved positions as compared to the Chlamydomonas reinhardtii 14-3-3 gene. D. salina 14-3-3 cDNA was inserted into the prokaryotic expression plasmid pET-28 and transformed into E. coli BL21, and the recombinant expressed 14-3-3 protein was purified from E. coli and immunized the rabbit. Indirect ELISA coated with 14-3-3 illustrated that the rabbit antisera titration was 1:1.00E + 06. Western blotting assays confirmed that prepared rabbit antibodies could recognize the recombinant 14-3-3 protein. Southern blotting results showed that there was only one copy of the 14-3-3 present in the genome of D. salina and 14-3-3 expression did not change throughout the Dnualiella cell cycle.

Fusion with matrix attachment regions enhances expression of recombinant protein in human HT-1080 cells.

Like endogenous proteins, recombinant foreign proteins produced in human cell lines also need post-translational modifications. However, high and long-term expression of a gene of interest (GOI) presents significant challenges for recombinant protein production in human cells. In this work, the effect of human matrix attachment region elements (MARs), including the ß-globin MAR (gMAR), chicken lysozyme MAR (cMAR), and a combination of these two, on the stable expression of GOI was assessed in human HT-1080 cells. After transfection with vectors containing the MAR elements and eGFP, stably HT-1080 cell pools were obtained under selective pressure. eGFP protein expression was analyzed by flow cytometry, while transgene copy number and eGFP mRNA expression levels were determined with qPCR and qRT-PCR technology. We found that MARs could not enhance transfection efficiency, but gMAR could significantly increase eGFP expression in stable HT-1080 cell pools by approximately 2.69-fold. Moreover, gMAR could also increase eGFP expression stability during long-term culture. Lastly, we showed that the effect of the MARs on transgenes was related to the gene copy number. In summary, this study found that MARs could both enhance the transgene expression and stability in HT-1080 cells.

Preparation of PU/Fibrin Vascular Scaffold with Good Biomechanical Properties and Evaluation of Its Performance in vitro and in vivo.

PURPOSE: The development of tissue-engineered blood vessels provides a new source of donors for coronary artery bypass grafting and peripheral blood vessel transplantation. Fibrin fiber has good biocompatibility and is an ideal tissue engineering vascular scaffold, but its mechanical property needs improvement. METHODS: We mixed polyurethane (PU) and fibrin to prepare the PU/fibrin vascular scaffolds by using electrospinning technology in order to enhance the mechanical properties of fibrin scaffold. We investigated the morphological, mechanical strength, hydrophilicity, degradation, blood and cell compatibility of PU/fibrin (0:100), PU/fibrin (5:95), PU/fibrin (15:85) and PU/fibrin (25:75) vascular scaffolds. Based on the results in vitro, PU/fibrin (15:85) was selected for transplantation in vivo to repair vascular defects, and the extracellular matrix formation, vascular remodeling, and immune response were evaluated. RESULTS: The results indicated that the fiber diameter of the PU/fibrin (15:85) scaffold was about 712nm. With the increase of PU content, the mechanical strength of the composite scaffolds increased, however, the degradation rate decreased gradually. The PU/fibrin scaffold showed good hydrophilicity and hemocompatibility. PU/fibrin (15:85) vascular scaffold could promote the adhesion and proliferation of mesenchymal stromal cells (MSCs). Quantitative RT-PCR experimental results showed that the expression of collagen, survivin and vimentin genes in PU/fibrin (15:85) was higher than that in PU/fibrin (25:75). The results in vivo indicated the mechanical properties and compliance of PU/fibrin grafts could meet clinical requirements and the proportion of thrombosis or occlusion was significantly lower. The graft showed strong vasomotor response, and the smooth muscle cells, endothelial cells, and ECM deposition of the neoartery were comparable to that of native artery after 3 months. At 3 months, the amount of macrophages in PU/fibrin grafts was significantly lower, and the secretion of pro-inflammatory and anti-inflammatory cytokines decreased. CONCLUSION: PU/fibrin (15:85) vascular scaffolds had great potential to be used as small-diameter tissue engineering blood vessels.

Retraction Note: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells.

This paper has been retracted at the request of the authors.

Pitavastatin up-regulates eNOS production by suppressing miR-155 expression in lipopolysaccharide-stimulated human umbilical vein endothelial cells.

AIM: Pitavastatin (Pit) has been proved to efficiently inhibit the onset and progression of atherosclerosis. However, the mechanism by which Pit exerts nonlipid-related effects, such as antiinflammatory actions, is not quite clear. Our study aimed at investigating the effect of Pit on the expression of endothelial NO synthase (eNOS) and miR-155 in LPS-stimulated HUVECs to reveal the antiinflammatory mechanism of pitavastatin. METHODS: HUVECs were isolated from newborn umbilical cords and used in the experiments at passages 2-5. Cells were treated with LPS (0.05, 0.1, 1 µg/L) or LPS (0.1 µg/L)+Pit (0.01, 0.1, 1 µmol/L), untreated cells were used as control. For LPS+Pit induction, cells were firstly incubated with Pit for 1 hour before coincubation with LPS for 24 hours. eNOS mRNA and miR-155 were detected by RT-PCR, and Western blotting was used to detect protein expression of eNOS. RESULTS: Treatment of HUVECs with LPS enhanced the expression of miR-155 and reduced the expression of eNOS in mRNA and protein level in a dose-dependent manner as revealed by RT-PCR and Western blotting, respectively. Pitavastatin ameliorated LPS-induced endothelial dysfunction through upregulation of eNOS expression and downregulation of miR-155 expression. CONCLUSION: Pitavastatin increases eNOS expression and inhibits of LPS-induced miR-155 expression.

Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells.

In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.